Simultaneous detection of herpes simplex virus types 1 and 2 by real-time PCR and Pyrosequencing

Abstract

Background:

Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions.

Objective:

To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype.

Study design:

ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5′ biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced.

Results:

The quantitative range of the assay extended from 10⁸ through 10⁰ copies of each virus (r² > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2.

Conclusions:

This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.

Introduction

Infections with herpes simplex virus (HSV) have reached epidemic proportions and continue to rise worldwide (Corey, 2002). Up to 80% of the American adult population has been infected with HSV type 1 (HSV-1), primarily during childhood (Smith and Robinson, 2002). The third National Health and Nutrition Examination Survey (NHANES III) performed from 1988 to 1994 reported the seroprevalence of HSV type 2 (HSV-2) in people 12 years and older in the United States to be 21.9%, or approximately 45 million people (Fleming et al., 1997). As many as one billion HSV-2 infections have been estimated worldwide (Cusini and Ghislanzoni, 2001). Although historically orolabial herpesvirus infections were epidemiologically associated with HSV-1 and anogenital herpes was almost exclusively attributed to HSV-2 infections, a recent shift in epidemiology has arisen (Mertz et al., 2003, Samra et al., 2003). A retrospective review of 499 college students at the University of Wisconsin–Madison revealed that the HSV-1 proportion of newly diagnosed genital herpes cases rose from 31% in 1993 to 78% in 2001 (Roberts et al., 2003). Internationally, HSV-1 was the predominant subtype of HSV encountered in genital specimens at the Beilinson Medical Center in Tel Aviv, Israel from 1993 through 2002 (Samra et al., 2003) and is responsible for 40% of all new genital cases in the United Kingdom (Scoular et al., 1990). This change has been attributed to a rise in oral-genital sexual contact that increases the opportunity for HSV-1 transmission, coupled with an escalation in condom usage in an effort to thwart the spread of HSV-2 and the human immunodeficiency virus (HIV) (Mertz et al., 2003).

Classical clinical symptoms of genital herpes caused by either subtype present as papules on the external genitalia, which form vesicular ulcers that scab and reepithelialize (Patel, 2002). Symptoms of non-classical infections, which can occur in up to 60% of genital herpes cases, include vulval/perianal fissures, perianal lesions, reddening of the buttocks and thighs, painful urination, vaginal and urethral discharges, and pain in the lower limbs and back (Cusini and Ghislanzoni, 2001). Although HSV-1 is increasingly the etiologic agent causing genital herpes, clinical outcomes differ depending on the infecting subtype as recurrent episodes have been reported to occur approximately five to fifteen times more frequently with HSV-2 than HSV-1 (Benedetti et al., 1994). Infection with HSV can modulate the susceptibility and severity of another sexually transmitted virus also, HIV. A scientific literature review by Fleming and Wasserheit (1999) reported that an HSV-induced disruption of the genital mucosal barrier could increase the risk of contracting HIV-1 up to three-fold. Also, HSV reactivation is proposed to enhance HIV-1 replication and retroviral shedding (Fleming and Wasserheit, 1999) and activated HSV-2 can be detected in HIV-1 positive patients three times more frequently than in non-infected individuals (Schacker et al., 1998).

Culture of HSV is time-consuming and although positive results are evident within one to three days of incubation, negative results cannot be confirmed for up to two weeks (Samra et al., 2003). Additionally, immunofluorescent analysis of a positive culture is required to differentiate between HSV-1 and HSV-2. Although the specificity of the viral culture is 100%, the sensitivity of this detection technique is highly dependent upon the stage of the infection in which the lesions are sampled and can be as low as 50% resulting in significant false negative results (Cusini and Ghislanzoni, 2001, Lafferty et al., 1987). Specimen shipping conditions from the collection site to the laboratory have also been reported to pronouncedly affect the capacity to successfully culture virus as was observed in a shift of PCR to viral culture positivity ratios from 3.8:1 during winter to 8.8:1 during summer months (Wald et al., 2003). Real-time PCR has emerged as a significantly more sensitive clinical diagnostic technique capable of providing accurate results in a fraction of the time of viral culture methods. A recent study which analyzed the presence of HSV in more than 36,000 mucosal secretions found the detection rate of HSV DNA by real-time PCR techniques to be 12.1% of the samples versus 3.0% detected by viral culture (Wald et al., 2003), but this assay did not discriminate between HSV-1 and HSV-2.

We report the development of a multiplex real-time PCR assay developed to simultaneously detect HSV-1 and/or HSV-2 on the Rotor-Gene 3000 platform. Sub-typing validation was performed by Pyrosequencing to obtain short sequences of nucleotides capable of identifying HSV-1 and/or HSV-2 DNA. This technique was applied to 4581 cervical swabs sampled from women primarily from six states and the prevalence of HSV detection in these specimens is discussed.